The present disclosure relates to a method for detecting lipid bilayer membrane particles or fragments thereof.
Hitherto, it has been known that cells release vesicles during apoptosis; however, it has been revealed that healthy cells also release vesicles similarly, and thus the physiological functions of the vesicles have attracted attention. Such extracellular vesicles (EVs) are derived from cells and thus have the form of particles covered with lipid bilayer membranes (lipid bilayer membrane particles; lipid vesicles), but it is found that there are several types of extracellular vesicles depending on derivations or characteristics thereof.
First, “apoptotic bodies (ABs)” are vesicles released from apoptotic cells, have a size of about 1000 to 4000 nm (1 to 4 μm) due to occurrence mechanism thereof, and serve functions such as induction of phagocytosis.
Meanwhile, both “microvesicles (MVs)” and “exosomes” are vesicles released from normal cells and discriminated from each other on the basis of the occurrence mechanism. Specifically, extracellular vesicles formed or secreted by sinking of membranes of multivesicular bodies (MVBs) in the inside thereof are defined as exosomes and the size thereof is about 30 to 200 nm. The exosomes contain a large number of Alix, Tsg101 and HSP70, which are endosome-binding proteins, and tetraspanin families (CD63, CD81, and CD9), which are membrane-spanning proteins, or the like. Thus, these proteins are used as exosome marker molecules in detection of exosomes in a sample derived from a living body. Further, regarding exosomes extracted using these molecules as markers, analysis of microRNA (miRNA) and the like contained therein has been conducted, and currently, comprehensive search tests for leading to cancer treatment have been advanced as a national project.
In contrast with the exosomes, extracellular vesicles budded directly from cell membranes (ectosomes) are defined as microvesicles (MVs). The microvesicles (MVs) are generated by fragmentation of a cell skeleton due to flip flop phenomenon of two occurrence mechanisms of microparticles (MPs), and thus the size thereof is about 100 to 1000 nm. The microvesicles contain a large number of proteins, nucleic acids, miRNAs, which are derived from cells (Annexin V, integrin, selectin, CD40 ligand, and metalloproteinase), and the like. These exosomes and MVs serve functions such as cell-to-cell communication. The ectosomes contain a large number of tissue factors (CD142: TFs), which are transmembrane proteins, and the like. Regarding markers of MVs or MPs, a surface antigen marker or the like of a parent cell serving as an expression origin is used together with size information and an activation marker, and for example, markers that are derived from vascular endothelial cells, monocytes, blood platelets, and the like have attracted attention.
Further, besides the aforementioned extracellular vesicles, circulating tumor cells (CTCs), circulating endothelial cells (CECs), and circulating endothelial progenitors (CEPs) are known as lipid vesicles that circulate in peripheral bloods in accordance with progression (metastasis or the like) of disease state of cancer. Since these are cells, similarly to the lipid vesicles, these cells have the form of particles covered with lipid bilayer membranes (lipid bilayer membrane particles).
The circulating tumor cells (CTCs) are defined as tumor cells that circulate in a peripheral blood flow of a cancer patient and are tumor cells that are invaded from a primary tumor or a metastatic tumor into blood vessels. Detection of the CTCs has recently attracted attention as one of methods for early detection of metastatic malignancy. The reason for this is that the detection of the CTCs is less invasive than detection using X-ray photography or a tumor marker in the blood serum and enables the diagnosis of metastatic malignancy to be precisely performed, and can be used as a barometer of prognostic prediction or treatment effect of patients.
Further, the circulating endothelial cells (CECs) are defined as mature cells peeled away from blood vessel walls due to natural metabolism of endothelial cells, and serve functions of vascularization and maintaining the blood vessel walls. It has been reported that the CECs increase in a large number of disease states of cardiovascular diseases, infective diseases, immunological diseases, tests after transplant, cancers, and the like. In particular, in cancer researches, the CECs are suggested as a non-invasive biomarker of angiogenic activity showing tumor regrowth, resistance to chemotherapy, early recurrence, and metastasis during or after chemotherapy. Meanwhile, it has been reported that the CEC level in healthy individuals is extremely low and is about 0.01 to 0.0001% of whole peripheral blood mononuclear cells.
Furthermore, it has been known that the circulating endothelial progenitors (CEPs) exist in peripheral blood mononuclear cell fractions of adults and the endothelial progenitors are locally grown and differentiated so that they involve in vascularization. The CEPs are recruited from the bone marrow even at the time of construction of new blood vessels in the case of cancers and are circulated in blood at the time of newly creating blood vessels according to metastasis of cancer. For these reasons, the CEPs are expected as a non-invasive biomarker similar to the CECs. Further, these CECs and CEPs are also studied for being used as a marker for determining the treatment effect of antibody drugs and presence/absence of adverse events.
All of the lipid bilayer membrane particles of extracellular vesicles, CTCs, CECs, CEPs, and the like described above are detected in blood circulating in a living body. For this reason, there is an advantage in that by detecting these lipid bilayer membrane particles in a blood sample derived from the living body, abnormity in the living body can be detected without performing an invasive biopsy test.
As a method for detecting such lipid bilayer membrane particles, conventionally, a substance which specifically binds to a surface antigen existing on surfaces of the lipid bilayer membrane particles (hereinafter referred to as “specifically-binding substance ”) such as an antibody or the like is caused to emit light using a fluorescence probe labeled with a fluorescent dye so as to be detected by flow cytometry or imaging cytometry (for example, see JP 2012-168012 A). Herein, the amount of the above-described lipid bilayer membrane particles existing in the sample is extremely scarce depending on circumstances. Further, for example, in the case of describing CTCs as an example, it is known that the lipid bilayer membrane particles exist in an amount corresponding to only about one cell of 109 to 1010 cells/mL of blood cells contained in blood of a metastatic cancer patient. In order to ensure the amount of the surface antigen for the purpose of enabling such a scarce detection target to be sufficiently measured, it is necessary to measure an extremely large number of particles (vesicles) also including particles (vesicles) serving as a noise source. However, since there is limitation on the number of measurable particles, a sufficient measurement number cannot be typically ensured, and thus, it is not possible to sufficiently increase detection sensitivity only by increasing the measurement number. For the purpose of condensing a scarce detection target in this way, a method has been known in which an antibody or the like that specifically binds to a detection target is conjugated with a magnetic bead, and then the detection target is condensed by a separation operation using a magnetic apparatus (a magnet) (for example, see JP 2013-015517 A).
Further, for the purpose of observing the forms of the lipid bilayer membrane particles (lipid vesicles) or observing the states of localized lipid (granules, cell vesicles, mitochondria, or the like) inside the particles (vesicles), a technique has been known in which a lipid bilayer membrane is stained using a fluorescent dye staining a lipid bilayer membrane (so-called lipid staining) (for example, see JP 2014-236685 A). Incidentally, a technique of using this lipid staining in combination with the aforementioned separation and detection method using flow cytometry or the like has not been known in the related art. For example, in Yusuke Yoshioka, Takahiro Ochiya, “Exosome provides new insight into liquid biopsy”, Cytometry Research, Japan Cytometry Society, Vol. 26 (2016) No. 1, pp. 1-6, a technique has been reported in which a protein existing on an exosome membrane is sandwiched by two types of monoclonal antibodies on which different modifications are conducted (one is a biotinylated antibody to which Alpha donor bead covered with streptavidin binds and the other is an antibody to which AlphaLISA acceptor bead is allowed to bind) (ExoScreen method). In this technique, only in a case where two types of antibodies come close within 200 nm, singlet oxygen generated by excitation of a photosensitizer contained in the donor bead undergoes chemiluminescence reaction with the acceptor bead to generate a fluorescence signal, so that the exosomes can be detected by measuring this fluorescence signal.